HIV-1 Gag

HIV-1 Gag
The HIV-1 Gag protein is the major structural protein required for virus assembly. It is synthesized as a polyprotein in the cytosol of an infected cell, and contains four functional segments; MA, CA (NTD and CTD), NC, and p6. The NC region is flanked by two "spacer" segments, denoted SP1 and SP2. The polyprotein is all alpha helical, except the NC region, which is composed of two RNA interacting zinc knuckle domains. Gag is often referred to as an "assembly machine" because expression of Gag alone is sufficient to produce budding virus-like particles (VLP's), due to multimerization of roughly 2000 Gag molecules per virion. Here, we will take a closer look at the MA, CA, and NC domains, and how the structural components of these domains aid in the assembly of virus particles. Viral particles can be classified as immature (pre-budding and non-infectious), and mature (post-budding and infectious), and this exchange is mediated by the HIV-1 protease. Upon viral budding, Gag is cleaved by the HIV-1 protease at multiple sites, thus possibly changing many of the structural interactions that make up the "immature" particle. For simplicity, we will only be discussing the immature formation of Gag on the plasma membrane of infected cells, as it coordinates organized viral budding. Also, it is thought that Gag forms a hexamer structure (deduced from electron microscopy studies) upon virus assembly, but because of the difficulties encountered by attempting to crystallize a multimeric structure, the exact formation of the hexamer is still up for debate. Current models predict that the MA and CA domains interact to form trimers, and these trimers conglomerate and display hexagonal symmetry.

Matrix (MA)
The MA domain (also called Matrix) is essential for proper targeting of Gag (and thus virus release) to distinct locations on the plasma membrane. The MA domain is myristylated post translationally, which is important for a stable association with the plasma membrane. Perhaps just as important for proper virus assembly, is the interaction of MA with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). It has long been known that HIV-1 budding occurs at sites rich in PI(4,5)P2 and cholesterol (lipid rafts, and tetraspanin enriched microdomains, TEMs), probably to ensure proper virus budding localized to the plasma membrane (elimination of PI(4,5)P2 causes virions to assembly at intracellular endosomes). More recently, structural analysis of MA has revealed a myristylation switch that allows exposer of the myristyl group to the plasma membrane only upon binding of MA to PI(4,5)P2, a mechanism ensuring proper localization of Gag to rafts within the plasma membrane.

When MA is not bound to PI(4,5)P2 (Figure 1), notice the alignment of helix 1, and more precisely, the orientation of Leu 8 and Glu 12(Show Residues ). In this PI(4,5)P2 unbound structure, the myristyl group is sequestered in the pocket of helix 1 created by Leu 8 and Glu 12. Upon binding of PI(4,5)P2 to the hydrophobic groove created by helix 2, a type 2 beta turn, and helix 5, a slight conformational switch occurs in helix 1 (Figure 2), causing a change in the alignment of Leu 8 and Glu 12,(Show Residues ) ejecting the myristyl group from it's sequestered state. This structural switch allows membrane anchoring to be directly coupled to proper membrane localization of Gag, and thus efficient particle release.

Capsid (CA)
 Once Gag is localized to discreet sites on the plasma membrane, multimerization of Gag takes place quite quickly, driven by the CA domain, and more specifically our focus here, the C-terminal domain of CA (CTD). There are four helices that contribute to the interaction of CA CTD with it's partner. A side-by-side interaction has been proposed (Figure 3), but many believe the forces involved in the side-by-side model are not great enough to account for the organization and structural stability of assembled Gag. Also, helix 1 of the CA CTD contains a very conserved region of residues within many retroviruses called the MHR (major homology region)MHR region. In the side-by-side model, the MHR is not responsible for the dimer organization, yet mutations in the MHR region have a greater impact on in vivo Gag assembly than mutations in the residues responsible for structural stablility of the side-by-side model. This led researchers to seek a domain swapped structure of the CA CTD. By making a single deletion of the Ala 177 residue (which lies in the loop between helix 1 and helix 2), the CA CTD domain adopts a domain-swapped conformation, in which the MHR of helix 1 is extended to contact helices 2,3, and 4 of the adjacent CA CTD domain (Figure 4)(MHR region ). Keep in mind these are only models of Gag dimerization and subsequent multimerization, and that precise interactions that take place during Gag assembly are yet to be agreed upon.

Note the CA NTD was not discussed here. Through biochemical studies, the NTD domain has been implicated in viral capsid assembly, but reliable structural data giving biological insights has been elusive.

Nucleocapsid (NC)


As we have seen, Gag is responsible for correct targeting of viral assembly to discreet sites on the plasma membrane, and viral capsid structure assembly into organized virus particles. However, Gag is also responsible for packaging of the viral RNA into budding virions, and this function in executed by the NC domain. The 5' LTR of HIV-1 genomic RNA contains a recognition element called the psi element. All retroviruses contain some type of psi element in order to get specific packaging of viral genomic RNA within the budding particles, and in the case of HIV-1, the psi element is 120 bases, and contains 4 stem-loop structures. Although the psi element can be quite variable, the SL3 loop is highly conserved within HIV-1 strains. Figure 5 shows the NC domain of HIV-1 NL4-3 in complex with the SL3 loop of the viral psi element. There are two zinc knuckle domains, with the zinc ion held by three Cys residues and a His(Knuckle ). Notice that guanine210 and guanine212 of the RNA interact with the F2 and F1 CCHC zinc knuckles respectively. In the F1 knuckle, G9 fits into a hydrophobic cleft formed by Val13, Phe16, Ile24, and Ala 25. G9 satisfies Watson-Crick hydrogen bonding by interacting with the NH groups from Phe16 and Ala25(Show residues ), and also the CO group of Lys14 (Show ). G7 interacts much the same with the F2 knuckle by hydrogen bonding with the NH groups of Trp37 and Met46, while also hydrogen bonding with the CO group of Gly35. The adenine211 nucleotide forms a hydrogen bond with a highly conserved Arg32 residue(Show ). Also, residues 3 thru 10 form a 3.10 helix, which nestles into the RNA major groove(Show helix ). These interactions allow viral RNA to be specifically packaged into virions. Also of note, it has been shown that NC binding of viral RNA increases the ability of Gag to multimerize, thus providing another mechanism to couple functional virion assembly to Gag multimerization and viral budding.

Additional Resources
For additional information, see: Human Immunodeficiency Virus